The present invention is directed to methods for decreasing the clearance rate of 1) vascular endothelial cell growth factor (hereinafter sometimes referred to as VEGF) and of 2) vascular endothelial cell growth factor particular variants, and further with respect to such variants, methods for preparing them and methods and compositions and assays utilizing them for producing pharmaceutically active materials having therapeutic properties not differing at least substantially in kind from the parent compound, VEGF, but having pharmacological properties that differ from the parent compound, VEGF. In particular, the assays using such variants can be employed to discover new materials having agonistic or antagonistic properties to VEGF.
The method employing VEGF and the VEGF variants hereof manifest a demonstrated slower clearance rate compared with native material so as to provide useful and perhaps safer alternatives for systemic administration, as lower doses are available because of such reduced clearance rates; hence, regimens of VEGF and VEGF variants provide longer availability for therapeutic effect.
VEGF is a naturally occurring compound that is produced in follicular or folliculo-stellate cells (FC), a morphologically well characterized population of granular cells. The FC are stellate cells that send cytoplasmic processes between secretory cells.
Several years ago a heparin-binding endothelial cell-growth factor called vascular endothelial growth factor (VEGF) was identified and purified from media conditioned by bovine pituitary follicular or folliculo-stellate cells. See Ferrara et al., Biophys. Res. Comm. 161, 851 (1989).
Although a vascular endothelial cell growth factor could be isolated and purified from natural sources for subsequent therapeutic use, the relatively low concentrations of the protein in FC and the high cost, both in terms of effort and expense, of recovering VEGF proved commercially unavailing. Accordingly, further efforts were undertaken to clone and express VEGF via recombinant DNA techniques. The embodiments of that research are set forth in the patent applications referred to supra; this research was also reported in the scientific literature in Laboratory Investigation 72, 615 (1995), and the references cited therein.
In those documents there is described an isolated nucleic acid sequence comprising a sequence that encodes a vascular endothelial cell growth factor having a molecular weight of about 45,000 daltons under non-reducing conditions and about 23,000 under reducing conditions as measured by SDS-PAGE. Both the DNA and amino acid sequences are set forth in figures forming a part of the present applicationxe2x80x94see infra.
VEGF prepared as described in the patent applications cited supra, is useful for treating conditions in which a selected action on the vascular endothelial cells, in the absence of excessive tissue growth, is important, for example, diabetic ulcers and vascular injuries resulting from trauma such as subcutaneous wounds. Being a vascular (artery and venus) endothelial cell growth factor, VEGF restores cells that are damaged, a process referred to as vasculogenesis, and stimulates the formulation of new vessels, a process referred to as angiogenesis.
VEGF is expressed in a variety of tissues as multiple homodimeric forms (121, 165, 189 and 206 amino acids per monomer) resulting from alternative RNA splicing. VEGF121 is a soluble mitogen that does not bind heparin; the longer forms of VEGF bind heparin with progressively higher affinity. The heparin-binding forms of VEGF can be cleaved in the carboxy terminus by plasmin to release (a) diffusible form(s) of VEGF. Amino acid sequencing of the carboxy terminal peptide identified after plasmin cleavage is Arg110-Ala111. Amino terminal xe2x80x9ccorexe2x80x9d protein, VEGF (1-110) isolated as a homodimer, binds neutralizing monoclonal antibodies (4.6.1 and 2E3) and soluble forms of FLT-1, KDR and FLK receptors with similar affinity compared to the intact VEGF165, homodimer.
VEGF contains a C-terminal heparin binding domain that generally spans the C-terminus beginning beyond about amino acid 120. Generally this domain carries a relatively large number of positively charged amino acids.
The present invention, inter alia, is predicated upon initial research results that compared the heparin binding properties of VEGF derived respectively from recombinant Chinese hamster ovary (CHO) and E. coli cells. This research resulted in the finding that the CHO-derived VEGF material contained various C-terminal xe2x80x9cprocessingxe2x80x9d resulting in forms having different lengths with respect to the heparin binding C-terminal domain versus the substantially full-length material derived via E. coli production. Such C-terminal processing includes internal clips, i.e., cleaved sites, within the heparin binding domain that may alter the secondary and tertiary structure of the heparin binding domain so as to decrease its affinity for heparin and endogenous heparan sulfate proteoglycans.
Further research indicated that such C-terminal processing of VEGF resulted in variants exhibiting slower rates of clearance and smaller volumes of distribution. Although these processed variants still possess at least a portion of the heparin binding domain, the modified domain bound heparin and heparin sulfate proteoglycans with lower affinity. The resultant effect was that less VEGF was cleared by non-specific target organs such as the liver.
It was therefore a further object of this research to produce VEGF variants that would have C-terminal variations with consequential varying heparin binding affinity resulting in variants of VEGF having a reduced clearance rate and hence longer retention within the body after systemic administration such that lower doses of the material were available for systemic administration for therapeutic effect.
In addition, further objective research produced results that indicate that heparin significantly alters the pharmacokinetics of VEGF in vivo, especially if heparin is coadministered with VEGF. This further research provided mechanistic proposals embodying both approaches to produce VEGF and VEGF variant regimens having the advantage of reduced clearance rates of the active principal(s), with consequential therapeutic benefit. The intellectual property of such research is the subject of the present invention.
Objects of this invention, as defined generally supra, are achieved in one aspect by the provision of vascular endothelial cell growth factor (VEGF) variants having modifications in the C-terminus heparin binding domain, said variants exhibiting reduced clearance rates for systemic administration generally at lower doses compared with native VEGF thus providing variants having longer availability for therapeutic effect.
In a preferred embodiment, such modifications result in structural alterations effected within the region of the C-terminus heparin binding domain bridging about amino acid 121 to about amino acid 165, and more preferably around the protease sensitive sites at positions 125 and 147 and/or at other sites within the domain where structural alterations alter functional binding characteristics, such as at the loci of positively charged amino acids.
The variants hereof may be prepared via recombinant DNA technology taking advantage of the available tools and techniques for providing DNA having deletions in the C-terminal domain such that the recombinant expression of such DNA provides VEGF variants wherein the C-terminus heparin binding domain contain deletions with resultant altered pharmacological properties affecting therapeutic results. Alternatively, such variants can be isolated from recombinant systems that cause proteolytic cleavage at the C-terminus (a largely basic amino acid containing domain) resulting in C-terminus heparin binding domain deletion variants. Alternatively, such C-terminus deletion variants are products of for example carboxypeptidase B treatment.
In other aspects, the present invention relates to DNA sequences encoding the various variants described supra, replicable expression vectors capable of expressing said DNA sequences via transforming DNA in a transformant host cell, and microorganisms and cell cultures which are transformed with such vectors.
In further aspects hereof, the present invention is directed to methods useful for the recombinant expression of such DNA referred to above including methods of isolation and purification as a part of recovery.
In yet further aspects, the present invention is directed to compositions useful for treating indications where vasculogenesis or angiogenesis is desired for treatment of an underlying disease state comprising a therapeutically effective amount of a VEGF variant hereof, advantageously being reduced in general dosage form because of the reduced clearance rates exhibited by the variants hereof, in admixture with a pharmaceutically acceptable carrier. Thus, the present invention provides variants of VEGF wherein their exhibited reduced rate of clearance provides resultant effects that provide in turn safer alternative systemic administration with lower doses resulting in longer availability for therapeutic effect. In addition, a decrease in potential toxic side effects would be expected due to the decrease in nonspecific binding of the heparin domain modified variants to non-target tissues.
Thus, the present invention is directed to VEGF C-terminal heparin binding domain variants having structural alterations that result in functional modification of the heparin binding characteristics of that VEGF variant molecule. Such structural alterations can be imparted by, for example, internal cleavage at various proteolytic sites and/or by various mutations. For example structural alterations may, and preferably do, direct a change in the ionic charge of the domain by replacement of the largely positively charged amino acids with negatively or neutrally charged amino acids and/or by other mutations resulting in derivatives such as by deletions of amino acids, substitutions, and so forth.
All such structural alterations are believed to result in molecules that have in turn altered confirmational structure that affects the heparin binding characteristics. It is believed that because the heparin binding domain is a highly positively charged domain, the binding characteristics with heparin are probably an ionic interaction. Therefore, structural alterations that would affect ionic interaction would in turn affect binding. Thus, any such structural alterations that result in affecting heparin binding are covered within the scope of the present invention, i.e., such VEGF variants hereof manifest functional effect. One major directive of this aspect of the invention is to reduce the overall positive charge manifest in the heparin binding domain of VEGF. Heparin sulfate proteoglycans exhibit an overall negative charge, and are associated with their progenitor vascular cells. Ionic binding of the proteoglycans with VEGF thus associates them with such vascular cells, or with red blood cells via a heparin embodied complex. Reduction of the overall positive charge of the heparin binding domain manifested by the VEGF variants hereof thus inhibits such binding resulting in such variants remaining in circulating plasma and removed from biological clearance mechanisms.
Expanding on the basic premise hereof based upon the finding of the effects of C-terminus heparin binding domain deletion variants of VEGF, the present invention is directed to all associated embodiments deriving therefrom, including recombinant DNA materials and processes for preparing such variants, materials and information for compounding such variants into pharmaceutically finished form and various assays using such variants.
The present invention is further premised on the finding that heparin significantly alters the pharmacokinetics of VEGF in vivo, especially if heparin is coadministered with VEGF.
It has been found that heparin impedes the distribution of VEGF to highly vascularized organs, for example the liver, a notorious clearance organ, by blocking the ionic binding interaction of VEGF with heparin sulfate proteoglycans. Thus, as discussed above with respect to the VEGF variants hereof, heparin serves to reduce the binding association of VEGF with vascular cells, or with red blood cells, freeing VEGF to remain in circulating plasma removed from biological clearance mechanisms.
Thus, coadministration, or at least co-presence of heparin with VEGF in vivo increases the VEGF concentration in plasma. Thus, VEGF exhibits high sustained concentration in plasma effecting reduced clearance rates.
This aspect of the present invention is thus directed to a method of reducing the clearance rate of VEGF in vivo comprising substantially coadministering VEGF with heparin and/or a heparin-like compound systemically.
Thus, in each of the major aspects of the present invention advantage is manifest in systemic administration of regimens of VEGF and/or VEGF variants whereby interference with the (natural) binding of the VEGF molecule with vasculature cells, for example as mediated by heparin sulfate proteoglycans increases distribution of the VEGF molecule in circulating plasma for consequential sustained therapeutic effect represented by reduced clearance rate(s).
In a generic iteration, the present invention is directed to a method of reducing the clearance rate of VEGF in vivo comprising substantially coadministering VEGF with heparin and/or a heparin-like compound systemically and of a VEGF variant hereof in vivo comprising administering said VEGF variant systemically.